Protocol for differentiating cardiomyocytes and generating engineered heart tissues from human feeder-free extended pluripotent stem cells

Summary

Extended pluripotent stem cells (EPSCs) possess a high differentiation capacity, potentially as a superior seed resource for generating cardiomyocytes. Here, we present a protocol for generating feeder-free EPSCs (ffEPSCs), cardiomyocytes, and engineered heart tissues (EHTs). We describe steps for converting human embryonic stem cells or induced pluripotent stem cells (ESCs/iPSCs) into ffEPSCs, followed by their long-term maintenance, cryopreservation, seed preservation, and differentiation into cardiomyocytes. We then detail procedures for constructing and culturing three-dimensional EHTs followed by their contraction force measurement and optical mapping.

For complete details on the use and execution of the protocol, please refer to Zheng et al.¹ and Li et al.²

Graphical abstract

Highlights

• •Instructions for converting human ESCs/iPSCs into ffEPSCs
• •Guidance on maintenance and differentiation into cardiomyocytes of ffEPSCs
• •Steps for the manufacture, cultivation, and functional evaluation of EHTs

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Extended pluripotent stem cells (EPSCs) possess a high differentiation capacity, potentially as a superior seed resource for generating cardiomyocytes. Here, we present a protocol for generating feeder-free EPSCs (ffEPSCs), cardiomyocytes, and engineered heart tissues (EHTs). We describe steps for converting human embryonic stem cells or induced pluripotent stem cells (ESCs/iPSCs) into ffEPSCs, followed by their long-term maintenance, cryopreservation, seed preservation, and differentiation into cardiomyocytes. We then detail procedures for constructing and culturing three-dimensional EHTs followed by their contraction force measurement and optical mapping.

Before you begin

The protocol below presents a detailed methodology for converting human embryonic stem cells or induced pluripotent stem cells (ESCs/iPSCs) into feeder-free extended pluripotent stem cells (ffEPSCs) and producing cardiomyocytes and engineering heart tissues. The protocol to generate ffEPSC-derived cardiomyocytes (EPSC-CMs) is of high efficiency and robustness and the EPSC-CMs exhibit improved mitochondrial function, calcium handling, and contractility properties at monolayer and micro-tissue levels. Prepare all reagents before starting the procedure. All procedures are performed under standard aseptic conditions using sterile equipment, sterile cell culture-grade reagents, and appropriate personal protective equipment.

Preparation of high-concentration small molecule stock solution

• 1.
  Preparation of Minocycline hydrochloride stock solution

  • a.
    Centrifuge the powder.
  • b.
    Add 5.062 mL of water to 50 mg of Minocycline Hydrochloride, dissolving it thoroughly to obtain a 20 mM stock solution.
  • c.
    Aliquot the stock solution into 20 μL portions.
  • d.
    Store at −20°C for 6 months.
• 2.
  Preparation of Human LIF Recombinant Protein stock solution

  • a.
    Centrifuge the powder.
  • b.
    Add 2.5 mL of 0.5% Bovine Serum Albumin (BSA) solution to 250 μg of Human LIF Recombinant Protein, dissolving it thoroughly to obtain a 100 μg/mL stock solution.
  • c.
    Aliquot the stock solution into 20 μL portions.
  • d.
    Store at −20°C for 6 months.
• 3.
  Preparation of (S)-(+)-Dimethindene maleate stock solution

  • a.
    Centrifuge the powder.
  • b.
    Add 1.224 mL of water to 10 mg of (S)-(+)-Dimethindene maleate, dissolving it thoroughly to obtain a 20 mM stock solution.
  • c.
    Aliquot the stock solution into 20 μL portions.
  • d.
    Store at −20°C for 6 months.
• 4.
  Preparation of Y-27632 stock solution

  • a.
    Centrifuge the powder.
  • b.
    Add 1.561 mL of DMSO to 10 mg of Y-27632 2HCl, dissolving it thoroughly to obtain a 20 mM stock solution.
  • c.
    Aliquot the stock solution into 20 μL portions.
  • d.
    Store at −20°C for no longer than 1 month or at −80°C for up to 1 year.

CRITICAL: DMSO is a low-toxicity organic solvent. Wear gloves and protective clothing while handling it in a biosafety cabinet. DMSO absorbs moisture and can reduce compound solubility. Use freshly opened DMSO whenever possible.

• 5.
  Preparation of IWR-1-endo stock solution

  • a.
    Centrifuge the powder.
  • b.
    Add 1.221 mL of DMSO to 5 mg of IWR-1-endo, dissolving it thoroughly to obtain a 10 mM stock solution.
  • c.
    Aliquot the stock solution into 20 μL portions.
  • d.
    Store at −20°C for no longer than 1 month or at −80°C for up to 1 year.
• 6.
  Preparation of CHIR-99021 stock solution

  • a.
    Centrifuge the powder.
  • b.
    Add 996.4 μL of DMSO to 5 mg of Laduviglusib (CHIR-99021) HCl, dissolving it thoroughly to obtain a 10 mM stock solution.
  • c.
    Aliquot the stock solution into 20 μL portions.
  • d.
    Store at −20°C for no longer than 1 month or at −80°C for up to 1 year.
• 7.
  Preparation of GSK126 stock solution

  • a.
    Centrifuge the powder.
  • b.
    Add 1.899 mL of DMSO to 5 mg of GSK126, dissolving it thoroughly to obtain a 5 mM stock solution.
  • c.
    Aliquot the stock solution into 20 μL portions.
  • d.
    Store at −20°C for no longer than 1 month or at −80°C for up to 1 year.
• 8.
  Preparation of 2-Deoxy-D-glucose stock solution

  • a.
    Weigh 1 g of 2-Deoxy-D-glucose.
  • b.
    Dissolve it in 15.229 mL of water to obtain a 400 mM stock solution.
  • c.
    Filter the solution using a 0.22 μm filter.
  • d.
    Aliquot the stock solution into 500 μL portions.
  • e.
    Store at −20°C for 6 months.
• 9.
  Preparation of IWP-2 stock solution

  • a.
    Centrifuge the powder.
  • b.
    Add 8.573 mL of DMSO to 10 mg of IWP-2, dissolving it thoroughly to obtain a 5 mM stock solution.
  • c.
    Aliquot the stock solution into 20 μL portions.
  • d.
    Store at −20°C for up to 1 year.
• 10.
  Preparation of DNaseI stock solution

  • a.
    Centrifuge the powder.
  • b.
    Add 5.5 mL ddH₂O to 110.0 mg DNase I, dissolving it thoroughly to obtain a 20 mg/mL stock solution.
  • c.
    Aliquot the stock solution into 50 μL portions.
  • d.
    Store at −20°C for up to 1 year.
• 11.
  Preparation of Thrombin stock solution

  • a.
    Centrifuge the powder.
  • b.
    Add 5.0 mL of 0.1% BSA to 250 IU thrombin, dissolving it thoroughly to obtain a 50 IU/mL stock solution.
  • c.
    Aliquot the stock solution into 40 μL portions.
  • d.
    Store at −20°C for 6 months.
• 12.
  Preparation of 6-Aminocaproic acid stock solution

  • a.
    Weigh 1.5 g of 6-Aminocaproic acid
  • b.
    Dissolve it in 30 mL ddH₂O to obtain a 50 mg/mL stock solution.
  • c.
    Filter the solution using a 0.22 μm filter.
  • d.
    Aliquot the stock solution into 1 mL portions.
  • e.
    Store at −20°C for up to 1 year.
• 13.
  Preparation of L-Ascorbic acid stock solution

  • a.
    Weigh 1.5 g of L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate.
  • b.
    Dissolve it in 30 mL ddH₂O to obtain a 50 mg/mL stock solution.
  • c.
    Filter the solution using a 0.22 μm filter.
  • d.
    Aliquot the stock solution into 200 μL portions.
  • e.
    Store at −20°C for up to 1 year.
• 14.
  Preparation of Collagenase Ⅰ stock solution

  • a.
    Add 100 mL FBS and 400 mL DPBS to 1 g Collagenase Ⅰ.
  • b.
    Dissolve it thoroughly to obtain a 2.0 mg/mL stock solution.
  • c.
    Aliquot the stock solution into 10 mL portions.
  • d.
    Store at −20°C for up to 1 year.
• 15.
  Preparation of 1-Thioglycerol stock solution

  • a.
    Add 100 μL of 45 mM 1-thioglycerol in 9.9 mL ddH₂O.
  • b.
    Dissolve it thoroughly to obtain a 0.45 mM stock solution.
  • c.
    Store at −20°C for up to 1 year.

CRITICAL: Gentle warming can dissolve it easily. Protect from prolonged exposure to light.

• 16.
  Preparation of Fibrinogen stock solution

  • a.
    Weigh 10 mg of fibrinogen.
  • b.
    Dissolve it in 1 mL DPBS (containing no Ca²⁺) to obtain a 10 mg/mL stock solution.
  • c.
    Filter the solution using a 0.22 μm filter.
  • d.
    Store at −20°C and ready to use.

CRITICAL: Prepare and use the hydrogel immediately to ensure its formation.

• 17.
  Preparation of Rhod-2 AM stock solution

  • a.
    Centrifuge the powder.
  • b.
    Dissolve rhod-2 AM in DMSO to prepare 2.5 mM stock solution.
  • c.
    Aliquot the stock solution into 1.0 μL.
  • d.
    Store at −20°C for up to 1 year.

CRITICAL: Protect from prolonged exposure to light.

CRITICAL: We recommend preparing all small molecules as high-concentration stock solutions before use. Avoid repeated freeze-thaw cycles.

Alternatives: CHIR-99021 and Y-27632 can be substituted with their non-salt forms.

The preparation of Matrigel-coated cell culture plates

Timing: 2 days

Note: Calculate the following amounts for a six-well cell culture plate. Adjust the usage amount based on the surface area of the culture dish used.

• 18.Place Matrigel in a 4°C refrigerator the day before to thaw slowly, and pre-cool disposable pipette tips and 1.5 mL centrifuge tubes at −20°C.
• 19.Pre-cool DPBS at 4°C the day before.
• 20.The next day, once the Matrigel has wholly thawed, aliquot it at 60 μL per 1.5 mL centrifuge tube. Operate on ice in a sterile environment, using pre-cooled materials. Store the aliquoted Matrigel at −20°C.

CRITICAL: Use the same batch of Matrigel validated for ESC culture. Aliquot large quantities of Matrigel into usable volumes and store at −20°C to prevent product condensation and ensure optimal performance.

• 21.Transfer 6 mL of pre-cooled DPBS buffer into a 15 mL tube using a pipette.
• 22.Pipette 1 mL of this solution into the container with 60 μL of Matrigel, gently mix, and transfer the mixture back into the 10 mL centrifuge tube, bringing the total volume to 6 mL, thus diluting the Matrigel to the working concentration (1%).

Note: Use the above formula for 1% Matrigel. For higher concentrations, adjust proportionally. For example, use 120 μL Matrigel in 6 mL DPBS for 2% concentration.

• 23.Add the diluted Matrigel solution to a six-well plate, 1 mL per well (∼1 mL/10 cm²).
• 24.Wrap the six-well cell culture plate with sealing film and store it in a 4°C refrigerator.
• 25.The matrigel-DPBS coating plate can be used after one day.

CRITICAL: Use the culture plates at least one day after Matrigel coating, and store them at 4°C for no more than one week.

Cell preparation: Culture of human ESC/iPSC lines

Timing: 5 days

• 26.Take the Matrigel-coated cell culture plate from the 4°C refrigerator and place it in a 37°C incubator to preheat for 30 min.
• 27.
  Thaw the frozen ESC/iPSC line.

  • a.
    Set the water bath to 37°C.
  • b.
    Retrieve one vial of ESC/iPSC from the liquid nitrogen tank and quickly transfer it to the water bath.
  • c.
    Gently shake the frozen vial to thaw the cells rapidly. Remove it when only a small ice crystal remains.
  • d.
    Prepare a 10 mL centrifuge tube in the biosafety cabinet.
  • e.
    Add 2 mL of DMEM/F-12 to the centrifuge tube.
  • f.
    Open the thawed vial and transfer all its contents to the centrifuge tube.
  • g.
    Centrifuge the tube at 200 g for 3 min.

CRITICAL: The liquid nitrogen tank is a hazardous device. Wear safety goggles and protective clothing when using it.

• 28.
  Seed ESC/iPSCs with an appropriate cell density.

  • a.
    Aspirate the supernatant from the centrifuge tube.
  • b.
    Add 1 mL of mTeSR1 to the centrifuge tube and resuspend the cells.
  • c.
    Take 10 μL of the cell suspension for counting.
  • d.
    In the biosafety cabinet, open the six-well cell culture plate prepared in step 1 and add 1 mL of mTeSR1 medium containing 10 μM Y-27632 to one well.
  • e.
    Plate 1.2×10⁵ ESC/iPSCs into the well.
  • f.
    Place the plate in a 37°C, 5% CO₂ incubator.

    Note: Y-27632 (10 μM) can be added to improve the cell state during passage.

• 29.
  After 24 h, replace the culture medium with fresh mTeSR1 Medium without Y-27632. Culture ESC/iPSCs to expand and restore their growth viability.

  • a.
    Replace the mTeSR1 medium every 24 h.
  • b.
    The cells should reach approximately 85% confluence in about 5 days.

    Note: An optimal initial state of ESC/iPSCs is crucial for subsequent procedures. We recommended that the recovered ESC/iPSCs should be cultured stably over two passages before ffEPSC induction.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Cell culture materials (medium, small molecules, growth factors)
mTeSR1	STEMCELL	Cat. No. 85850
DMEM/F-12	Gibco	Cat. No. C11330500BT
DPBS	Gibco	Cat. No. 14190144
KnockOut DMEM/F-12	Gibco	Cat. No. 12660012
Neurobasal medium	Gibco	Cat. No. 21103049
Matrigel	Corning	Cat. No. 354277
DMEM, no glucose, no glutamine, no phenol red	Gibco	Cat. No. A1443001
GlutaMAX	Gibco	Cat. No. 41090036
MEM Non-essential amino acids solution (NEAA)	Gibco	Cat. No.11140050
N-2 serum-free supplement, 100X	BasalMedia	Cat. No. S430J4
B-27 serum-free supplement, without VA, 50X	BasalMedia	Cat. No. S441J7
Knockout serum replacement	Gibco	Cat. No. 10828028
Penicillin-Streptomycin	Gibco	Cat. No. 15140-122
Accutase	Sigma	Cat. No. A6964
TrypLE Express	Gibco	Cat. No. 12604021
CS10	STEMCELL	Cat. No. 7930
2-Mercaptoethanol	Gibco	Cat. No. 21985023
Minocycline, hydrochloride	Santa Cruz	Cat. No. sc-203339
Human LIF recombinant protein	PeproTech	Cat. No. 300-05
(S)-(+)-Dimethindene maleate	Tocris	Cat. No.1425
Y-27632 2HCl	Selleck	Cat. No. S1049
IWR-1-endo	Selleck	Cat. No. S7086
Laduviglusib (CHIR-99021) HCl	Selleck	Cat. No. S2924
GSK126	Selleck	Cat. No. S7061
2-Deoxy-D-glucose	APExBIO	Cat. No. B1027
DMEM, low glucose, pyruvate	Gibco	Cat. No. 10567014
DMEM, high glucose, pyruvate	Gibco	Cat. No. C11995500BT
BASIC RPMI 1640 medium	Gibco	Cat. No. C11875500BT
DMEM, powder, low glucose, pyruvate	Gibco	Cat. No. 31600034
L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate	Sigma	Cat. No. A8960
6-Aminocaproic acid	Sigma	Cat. No. A2504
Collagenase Ⅰ from Clostridium histolyticum	Sigma	Cat. No. C0130
Trypsin-EDTA (0.25%), phenol red	Gibco	Cat. No. 25200056
DNase I, bovine pancreas	Millipore	Cat. No. 260913
Y-27632 (dihydrochloride)	STEMCELL	Cat. No. 72308
Fibrinogen from human plasma	Sigma	Cat. No. F3879
Thrombin from human plasma	Sigma	Cat. No. T6884
Vitamin B12	Sigma	Cat. No. V6629
B-27 supplement (-insulin)	BasalMedia	Cat. No. X064C5
Polydimethylsiloxane (PDMS)	Dow Corning	N/A
Sodium pyruvate	Gibco	Cat. No. 11360070
1-Thioglycerol	Sigma	Cat. No. M6145
Fluo-4, AM, cell-permeant	Invitrogen	Cat. No. F14201
Pluronic F-127	Sigma	Cat. No. P2443
IWP-2	Tocris	Cat. No. 3533
Rhod-2 AM	Thermo Fisher Scientific	Cat. No. R1245MP
Antibodies
Anti-DNMT3L antibody (1:200 dilution)	Abcam	Cat. No. ab194094
OCT-4 antibody (1:200 dilution)	Cell Signaling Technology	Cat. No. 2750S
PE mouse anti-cardiac troponin T (1:50 dilution)	BD Pharmingen	Cat. No. 564767
Anti-α-Actinin (Sarcomeric) (1:200 dilution)	Sigma	Cat. No. A7811
Equipment
Tissue culture-treated plate, 6-well	NEST	Cat. No. 703001
Tissue culture-treated plate, 12-well	NEST	Cat. No. 712001
Tissue culture-treated plate, 24-well	NEST	Cat. No. 702001
15-mL and 50-mL centrifuge tubes	NEST	Cat. No. 610001, 610101
10, 200, and 1,000 μL filter tips	Thermo QSP	Cat. No. 104-Q, 090-B-Q, 112NXL-Q
Handheld Pipetman	Gilson International	Cat. No. P20N, P200N, P1000N
0.22-μm Syringe filters	Biotool	Cat. No. B82011
Cryogenic vials	Nunc	Cat. No. 375418
Freezing container	Thermo Fisher Scientific	N/A
−80°C freezer	Panasonic	Cat. No. MDF-U54V
Liquid nitrogen tank	Thermo Fisher Scientific	Cat. No. Locator 4
37°C, 5% CO2 cell-culture incubator	Panasonic	Cat. No. MCO-18AC
Biological safety cabinet/laminar flow hood	Hadonglian	Cat. No. BSC-1100LIIA2
Water bath	Yiheng	Cat. No. HWS-12
Inverted phase-contrast fluorescence microscope	Olympus	Cat. No. IX53
Electrical stimulator	Chengdu Instrument Factory	Cat. No. YC-2
Optical Mapping	SciMedia	N/A
Software and algorithms
OMapScope5	MappingLab	https://mappinglab.com/product/omapscope-analysis
GraphPad Prism (8.0.2)	GraphPad	https://www.graphpad-prism.cn/?c=i&a=prism

Materials and equipment

Reagents for cell culture

ffEPSC medium
Reagent	Stock concentration	Final concentration	Amount
Neurobasal Medium	N/A	N/A	22.56 mL
KnockOut DMEM/F-12	N/A	N/A	22.56 mL
Knockout Serum Replacement	N/A	5%	2.5 mL
GlutaMAX Supplement	N/A	1%	500 μL
Non-Essential Amino Acids Solution	N/A	1%	500 μL
Penicillin-Streptomycin	N/A	1%	500 μL
B-27 Serum-Free Supplement liquid	50×	0.5×	500 μL
N-2 Supplement liquid	100×	0.5×	250 μL
2-Mercaptoethanol	55 mM	0.11 mM	100 μL
Human LIF Recombinant Protein	100 μg/mL	10 ng/mL	5 μL
Minocycline	20 mM	2 μM	5 μL
(S)-(+)-Dimethindene maleate	20 mM	2 μM	5 μL
Y-27632 2HCl	20 mM	2 μM	5 μL
IWR-1-endo	10 mM	1 μM	5 μL
Laduviglusib (CHIR-99021) HCl	10 mM	1 μM	5 μL
Total	N/A	N/A	50 mL

Store at 4°C for up to 2 weeks. Pre-warm the culture medium to 37°C before use.

CRITICAL: 2-Mercaptoethanol is toxic and can cause severe eye damage and skin irritation. Wear gloves and protective clothing, and follow all necessary safety precautions.

Alternatives: the glycolysis inhibitor Aurintricarboxylic acid (ATA) (50 μM) can be used instead of 2-Deoxy-D-glucose.

ffEPSC cleaning buffer

Reagent	Final concentration	Amount
KnockOut DMEM/F-12	N/A	23.75 mL
Neurobasal Medium	N/A	23.75 mL
Knockout Serum Replacement	5%	2.5 mL
Total	N/A	50 mL

Store at 4°C for up to 2 weeks.

ffEPSC accelerated conversion medium

ffEPSC medium supplemented with 1 μM GSK126.

ffEPSC long-term maintenance medium

ffEPSC medium supplemented with 2 mM 2-Deoxy-D-glucose

Differentiation medium

Reagent	Final concentration	Amount
BASIC RPMI 1640 Medium	N/A	48.8 mL
B-27 supplement (-insulin) 10×	1×	1 mL
L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate	200 μg/mL	200 μL
Total	N/A	50 mL

Store at 4°C for up to 2 weeks.

Trypsin neutralizing medium

Reagent	Final concentration	Amount
DMEM, High Glucose, Pyruvate	N/A	2.495 mL
FBS	N/A	2.5 mL
DNase I	20 μg/mL	5 μL
Total	N/A	5 mL

Ready to use.

2×Low glucose DMEM

Reagent	Final concentration	Amount
DMEM, powder, low glucose, pyruvate	N/A	1 package
NaHCO3	N/A	3.024 g
ddH2O	N/A	N/A
Total	N/A	500 mL

Adjust pH to 7.6. Filter the solution using a 0.22 μm filter. Aliquot the medium into 40 mL portions and store at −20°C for up to 1 year.

1×Cardiac Medium

Reagent	Final concentration	Amount
DMEM, low glucose, GlutaMAX Supplement, pyruvate	N/A	35.18 mL
FBS	10%	4 mL
Vitamin-b12	2 μg/mL	20 μL
6-Aminocaproic acid	2 mg/mL	800 μL
Total	N/A	40 mL

Store at 4°C for up to 2 weeks.

2×Cardiac Medium

Reagent	Final concentration	Amount
2×DMEM, low glucose	N/A	30.36 mL
FBS	20%	8 mL
Vitamin-B12	4 μg/mL	40 μL
6-Aminocaproic acid	4 mg/mL	1.6 mL
Total	N/A	40 mL

Store at 4°C for up to 2 weeks.

Early cardiac medium

Reagent	Final concentration	Amount
BASIC RPMI 1640 Medium	N/A	22.6 mL
DMEM, no glucose, no glutamine, no phenol red	N/A	22.6 mL
6-Aminocaproic acid	2 mg/mL	2 mL
B-27 supplement (-insulin)	1×	1 mL
GlutaMAX Supplement	1%	500 μL
Sodium Pyruvate	1%	500 μL
Non-essential amino acids	1%	500 μL
L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate	200 μg/mL	200 μL
Lactate	4 μM	50 μL
1-Thioglycerol	0.45 mM	50 μL
Total	N/A	50 mL

Store at 4°C for up to 2 weeks.

Late cardiac medium

Reagent	Final concentration	Amount
DMEM, low glucose, GlutaMAX Supplement, pyruvate	N/A	44.75 mL
FBS	5%	2.5 mL
6-Aminocaproic acid	2 mg/mL	2 mL
Non-essential amino acids	1%	500 μL
L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate	200 μg/mL	200 μL
1-Thioglycerol	0.45 mM	50 μL
Total	N/A	50.0 mL

Store at 4°C for up to 2 weeks.

Tyrode’s solution

Reagent	Final concentration	Amount
NaCl	135 mM	3.944 g
KCl	5.4 mM	0.201 g
CaCl2	1.8 mM	0.100 g
MgCl2	1 mM	0.047 g
NaH2PO4	0.33 mM	0.020 g
Glucose	5 mM	0.450 g
HEPES	5 mM	2.5 mL
ddH2O	N/A	N/A
Total	N/A	500 mL

Adjust pH to 7.4 and filter through 0.22 μm filter. Store at 4°C for up to 2 weeks.

Step-by-step method details

Generation of ffEPSCs

Timing: 5 days

This protocol applies to human ESCs/iPSCs, including the verified conversion of HUES8, H9, PGP1 and WTC into ffEPSCs.

• 1.Prepare the cell culture plate. Prepare 6-well plates coated with 1% and 2% Matrigel a day before, following the same procedure.
• 2.
  Replate ESCs/iPSCs in active growth.

  • a.
    Discard the old medium from the ESCs/iPSCs.
  • b.
    Wash once with DPBS.
  • c.
    Add 1 mL Accutase.
  • d.
    Place the cell culture plate in the 37°C incubator for 3 min.
  • e.
    Remove the plate and observe under a microscope until the cells become rounded, indicating complete digestion.
  • f.
    Add 1 mL DMEM/F-12, resuspend the cells, and transfer to a centrifuge tube.
  • g.
    Centrifuge the tube at 200 g for 3 min.
  • h.
    Discard the supernatant and retain the bottom cells.
  • i.
    Add 1 mL of mTeSR1 to the centrifuge tube and resuspend the cells.
  • j.
    Take 10 μL of the cell suspension for counting.
  • k.
    2.4×10⁵ (2.3×10⁴/cm²) ESCs/iPSCs were inoculated into a 6-well plate coated with Matrigel (1%).
  • l.
    Culture ESCs/iPSCs with mTeSR1 for 24–48 h.
• 3.
  After 1–2 days, the ESCs/iPSCs will form small colonies and begin ffEPSC conversion.

  • a.
    Aspirate the old medium.
  • b.
    Wash twice with ffEPSC cleaning buffer.
  • c.
    Add 2 mL ffEPSC medium to each well of the 6-well plate.
• 4.
  After another 1–2 days in the ffEPSC medium, the cell confluence should reach ∼80%, and the cells will be ready for passaging.

  Note: After 24 h in ffEPSC medium, the cell morphology changes from the compact, three-dimensional ESC/iPSC colonies to a more spread-out, loose intermediate state. During this process, cell apoptosis is negligible.

  • a.
    Aspirate the old medium.
  • b.
    Wash once with ffEPSC cleaning buffer.
  • c.
    Incubate with 1 mL of TrypLE at 37°C for 1–2 min to dissociate the cells into small colonies.

    CRITICAL: The digestion time needs to be carefully controlled. After 1 min of digestion, observe the progress under a microscope and promptly stop the reaction.

  • d.
    Add 1 mL of ffEPSC cleaning buffer to each well to terminate the digestion.
  • e.
    Gently pipette the cells and transfer the cell suspension to a 10 mL centrifuge tube.
  • f.
    Collect the cells by centrifuging at 200 g for 3 min.
  • g.
    Discard the supernatant and resuspend the cells in the ffEPSC medium.
  • h.
    Discard the DPBS from the 6-well cell culture plate coated with 2% Matrigel and add 2 mL ffEPSC medium supplemented with 1 μM GSK126.
  • i.
    Seed small colonies at a 1:4 to 1:5 ratio (approximately 3×10⁵ cells).
  • j.
    Incubate the cells in a 37°C, 5% CO₂ incubator.

    Note: During the initial days, a selection process occurs, and significant cell death is expected.

• 5.
  Maintain ffEPSCs

  • a.
    Wash once daily with EPSC cleaning buffer.
  • b.
    Replace with fresh ffEPSC medium containing GSK126.
  • c.
    After approximately 3–4 days, the cells should reach 80% confluence, and the dome morphology will begin to appear.

Long-term maintenance of ffEPSCs

Timing: 10 days to infinity

The first-generation ffEPSCs are above 80% confluency and are ready for digestion and passage. Subsequent multiple passages help stabilize the state of ffEPSCs, and additionally, long-term maintenance culture can be carried out for over 100 passages.

• 6.Remove the old medium and wash it once with the ffEPSC cleaning buffer.
• 7.Add 1 mL TrypLE and digest at 37°C for 5 min.
• 8.Add 1 mL ffEPSC cleaning buffer to stop digestion and gently pipette cells off.
• 9.Centrifuge at 200 g for 3 min.

CRITICAL: As the culture progresses, the ffEPSC morphology becomes more compact. Use TrypLE for all subsequent ffEPSC digestions to facilitate quick dissociation.

• 10.Discard the supernatant and resuspend cells in the ffEPSC medium. Passage cells at a ratio of 1:4-1:5. For passages P1-P5, the ffEPSC medium includes 1 μM GSK126.

Note: GSK126 aids in the ffEPSC transformation process and is optional.

• 11.Cells typically reach 80% confluency in 3–4 days and can be passaged again following the same procedure.
• 12.After 5 consecutive passages, ffEPSCs stabilize and can be characterized and maintained.
• 13.Remove GSK126 from the ffEPSC medium during extended maintenance and switch to 2 mM 2-Deoxy-D-glucose. Change the medium daily.

Note: Inhibiting glycolysis helps maintain the ffEPSC state in the long term.

• 14.ffEPSC can be continuously cultured beyond 100 passages.

CRITICAL: Prolonged in vitro maintenance of ffEPSCs is generally not recommended as it might lead to genomic instability.

Cryopreservation and seed preservation of ffEPSCs

Timing: 30 min

After five generations of ffEPSC culture, the cells tend to stabilize and can be cryopreserved for long-term storage. We recommend freezing clones with a regular dome shape that have passed ffEPSC detection criteria to be saved as seed cells for the following differentiation.

• 15.
  Digest and collect ffEPSCs.

  • a.
    Remove the old medium and wash once with DPBS.
  • b.
    Add 1 mL TrypLE and incubate at 37°C for 5 min for cell dissociation.
  • c.
    Add 1 mL of ffEPSC cleaning buffer to resuspend the cells.
  • d.
    Transfer to a 10 mL centrifuge tube and centrifuge at 200 g for 3 min.
  • e.
    Discard the supernatant.
  • f.
    Add 1 mL of DPBS to resuspend the cells.
  • g.
    Count the cells using a 10 μL sample.
  • h.
    Centrifuge the remaining cells again under the same conditions.
• 16.Discard the supernatant and resuspend the cell pellet in 1 mL of CS10 for every 1×10⁶ cells, then transfer them to cryovials.
• 17.Label each vial with crucial information such as cell generation, number, and date.

CRITICAL: The number of cells to be cryopreserved should be at least 8×10⁴, as we assume massive cell death during the freezing process. If too few cells are frozen, successful recovery may not be possible.

• 18.Transfer the cryovials to a gradient cooling freezing container and place the freezing container in a −80°C freezer.
• 19.After 2 days, the cryovials are quickly transferred to a liquid nitrogen tank for long-term storage.

Differentiation of ffEPSCs into cardiomyocytes

Timing: 17 days

The starting cells must be undifferentiated primarily to ensure high differentiation efficiency.

• 20.Before thawing, prepare a new tissue culture dish pre-coated with 1% Matrigel.
• 21.To passage ffEPSCs, plate 3×10⁴ cells per cm² on 1% Matrigel-coated glass coverslips with ffEPSC medium.
• 22.24 h later (Day 1), aspirate the culture medium and wash the cell once with DPBS. Add 1.5 mL mTeSR1 per well of 6-well plate.
• 23.Incubate the plate in the incubator and change the fresh mTeSR1 medium every 24 h.
• 24.48 h later (Day 3), treat the cells with 50–90% confluence with 5–7.5 μM CHIR-99021 in the differentiation medium.

CRITICAL: The proper confluency must be checked under a microscope to ensure high differentiation efficiency.

• 25.48 h later (Day 5), replace the cell culture medium with the differentiation medium.
• 26.24 h later (Day 6), treat the cells with 5 μM IWR-1 and 2.5 μM IWP-2 in the differentiation medium.
• 27.48 h later (Day 8), change the differentiation medium containing no IWR-1 or IWP-2 every two days.
• 28.The beating cardiomyocytes usually appear around day 10, and the generated cardiomyocytes will be isolated for further use before day 17.

Construction and culture of three-dimensional EHTs

Timing: 14 days

• 29.
  Preparation of mold

  • a.
    To generate three-dimensional CM-derived EHTs, prepare the PDMS mold with dimensions of 8.0 mm × 1.5 mm × 1.5 mm (length × width × height) as described.³
  • b.
    Cut the nylon paper into a square frame, which is typically described as 0.15 mm thick and 1.5 mm wide on each side.

    CRITICAL: PDMS molds are coated with 0.2% (wt/vol) solution of pluronic F-127 to prevent hydrogel adhesion in advance.

  • c.
    Place 1× cardiac culture medium, 2× cardiac culture medium, Matrigel, fibrinogen (10 mg/mL), and thrombin (50 IU/mL) stock solutions on ice.
  • d.
    Dry PDMS molds with a nitrogen gun and place the modes in the 12-well tissue culture plate.
  • e.
    Fix the nylon frame to the mold with pins for further use. The nylon frame should fully contact the PDMS base.

    CRITICAL: The treated modes and frames need to be used in 24 h.

• 30.
  Isolation of ffEPSC-derived cardiomyocyte

  • a.
    After the differentiation from ffEPSCs for 15–17 days, the cardiomyocytes were ready to use in further process.
  • b.
    Aspirate the culture medium and wash the ffEPSC-derived cardiomyocytes with DPBS. Add 1.5 mL Collagenase I per well of 6-well plates.
  • c.
    Place the 6-well plate in an incubator (37°C, 5% CO₂) for 45 min.
  • d.
    Collect the cell clusters with the Collagenase I solution in the 15 mL centrifuge tube.

    CRITICAL: The cell density should be no more than 1×10⁷ cells/mL to ensure digestion efficiency.

  • e.
    Centrifuge at 200 g for 3 min at 25°C, and then carefully remove the supernatant.
  • f.
    Resuspend the cell pellet with 5 mL high-glucose DMEM.
  • g.
    Repeat the step e.
  • h.
    Add 1 mL trypsin solution and gently resuspend the cell pellet.
  • i.
    Quickly shake the 15 mL centrifuge tube for 4–5 min in 37°C water bath to disperse the cell clusters.

    CRITICAL: Prevent cell exposure to trypsin solution for extended periods (≤5 min).

  • j.
    Once the suspension is cloudy with no prominent cell clumps, add 2 mL of trypsin neutralizing medium to deactivate trypsin.
  • k.
    Determine the cell density of viable cells using a hemocytometer and trypan blue exclusion or automated cell counter.
  • l.
    Centrifuge at 200 g for 3 min at 25°C. After removing the supernatant, add 1× cardiac medium to prepare the cell mixture in a final concentration of 43.2 μL per million cells.
• 31.
  Cross-linking of Hydrogel

  • a.
    Aliquot the cardiomyocyte’s suspension into 43.2 μL portions and place them on ice.
  • b.
    Prepare the hydrogel mixture by adding 2× cardiac medium, Matrigel, and fibrinogen solutions at a 2:1:2 volume ratio in the 1.5 mL tube as Solution A.
  • c.
    Add 1.8 μL thrombin stock solution to the cell suspension as Solution B.

    CRITICAL: Ensure Solution B is mixed well to prevent heterogeneous tissue structure.

  • d.
    Mix 45.0 μL Solution A with 45.0 μL Solution B and quickly inject it into the PDMS molds. Each EHT contained 5×10⁵ cells in a 45 μL volume.

    CRITICAL: Cell–hydrogel mixture will polymerize within 5 min at 25°C.

  • e.
    Place the molds with EHTs in an incubator (37°C, 5% CO₂) for 30 min.
  • f.
    Remove the pins, and gently peel the EHT-attached nylon frame from the PDMS mold. Place it in the 12-well plate with 1.5 mL early cardiac medium (with 10 μM Y-27632 and 10% FBS for 24 h).
• 32.
  Culturing of three-dimensional EHTs

  • a.
    Change the culture medium every other day and place it on a plate rocker with 0.5 Hz rocking speed.

    CRITICAL: EHTs must culture on a plate shaker with a left-right tilt angle greater than 15 degrees.

  • b.
    Change the medium to a late cardiac medium from day 6. After 14 days of cultivation, assess EHTs' relevant functions.

Contraction force measurement of EHTs

Timing: 45 min

• 33.Turn on the force measurement equipment and load the stretch program.
• 34.Calibrate the device and switch to data acquisition mode to preheat the instrument for 30 min.

CRITICAL: After the force transducer is stable, proceed to the next step of the experiment.

• 35.Add pre-warmed Tyrode’s solution to the tissue bath and maintain at 37°C.
• 36.Place the EHT into the tissue bath. Pin the two sides of the EHT between the holder and the force sensor, respectively.
• 37.Adjust the EHT to the original length and cut the side nylon frame at the center.
• 38.Record the spontaneous contraction force of the EHT for 2 min.
• 39.Stimulate the EHT at 10 V electrical pulse for the pacing frequencies: 0, 0.5 Hz, 1 Hz, 1.5 Hz, 2 Hz, 2.5 Hz, 3 Hz, 3.5 Hz, and 4 Hz. Each frequency should be maintained for at least 20 s and recorded for 5–10 s each recording.
• 40.Gradually stretch the EHT bundle from 0% to 20% beyond the original length in 2% increments using the linear actuator while continuing 2 Hz electrical stimulation. Record data for 40 s at each stretch length.
• 41.Stop recording and save the contraction force data.
• 42.Remove the EHT and discard the used Tyrode’s solution.
• 43.Repeat steps 35–42 for the next test.
• 44.Analyze the recorded contraction force data using a customized program.

Optical mapping of EHTs

Timing: 45 min

• 45.Turn on and initialize the optical mapping device. Preheat the device for 30 min.
• 46.Add pre-warmed Tyrode’s solution to the tissue bath and maintain it at 37°C.
• 47.Prepare a 2.5 μM Rhod-2 AM solution with the high glucose DMEM. Add the solution to a 12-well plate.
• 48.Aspirate the culture medium from the EHT, then wash the EHT once with Tyrode’s solution.
• 49.Incubate the EHT into the Rhod-2 AM solution at 37°C for 25 min.
• 50.Wash the EHT twice with Tyrode’s solution, then incubate the EHT in the culture medium containing 2.5 μM Blebbistatin at 37°C for 10 min to inhibit contraction-reduced motion artifacts during recording.
• 51.Transfer the EHT into the tissue bath.
• 52.Switch the optical mapping device camera to preview mode and adjust the EHT to the center of the view.
• 53.Place the point electrode at the corner of the EHT.
• 54.Record 10 s of electrical activity induced by a 1 Hz electrical pulse (10 V) applied via the point electrode. Use a 594-channel photodiode array in object (1.6×) mode for recording.

CRITICAL: Before recording complete electrical activity, 5 s of pre-electrical stimulation is applied to ensure EHT response to pacing frequency.

• 55.Stop recording and save the optical mapping data.
• 56.Analyze the conduction velocity (CV) data using BV Ana software and process the action potential duration (APD) data using OMapScope software.

Expected outcomes

Characterization of ffEPSCs

Morphologically, ffEPSCs grow as small dome-shaped colonies with diameters ranging from 10 to 80 μm. These colonies are densely packed, have smooth surfaces, and exhibit a three-dimensional structure. Qualified ffEPSC morphology is one of the criteria for assessing their status. At the gene expression level, ffEPSCs display a more pronounced expression of naive pluripotency gene clusters. While expressing classical pluripotency markers such as OCT4 and NANOG, they also show higher levels of pre-implantation embryonic genes like DNMT3L and KLF4, and almost no expression of post-implantation embryonic genes such as DUSP6 and ZIC2. Additionally, immunofluorescence can detect OCT4 proteins, with DNMT3L serving as a specific marker for ffEPSCs compared to ESCs/iPSCs (Figure 1).

Figure 1.

Conversion process and representative makers for ffEPSCs

(A) A schematic representation of the conversion of human ESCs/iPSCs to ffEPSCs shows the morphological changes the cells undergo. The final ffEPSCs form small dome-shaped colonies. Scale bar, 100 μm.

(B) Immunofluorescence staining of ffEPSC colonies. Both ESCs/iPSCs and ffEPSCs express OCT4, but only ffEPSCs express DNMT3L. Scale bar,100 μm.

(C) At the RNA level, three sets of markers are tested to reflect the completion of ffEPSC conversion. This is characterized by a slight downregulation of classic pluripotency genes OCT4 and NANOG, a significant decrease in post-implantation genes DUSP6 and ZIC2, and a dramatic upregulation of pre-implantation genes DNMT3L and KLF4. n = 3 for each group. Data are represented as mean ± SEM.∗∗p < 0.01, ∗∗∗p < 0.001.

For further characterization, karyotype analysis, together with assays such as teratoma formation and embryo chimerism, are recommended.

High-efficiency ffEPSC-derived cardiomyocytes

The simple protocol is able to induce ffEPSCs to cardiomyocytes in 17 days and further generate beating cardiomyocytes in as few as 8 days (Figure 2). More importantly, the cardiomyocytes differentiated by this protocol show high stability among different batches. More than 80% of these cells display positive cTnT estimated by flow cytometry. An average of 2 million cells can be harvested from a single well of a 12-well plate. Those cardiomyocytes can be used for cardiac disease modeling and drug screening.

Figure 2.

Cardiomyocytes differentiation process from ffEPSCs and characteristic of EHTs

(A) A schematic representation of ffEPSC differentiation into cardiomyocytes shows the morphological changes the cells undergo.Scale bar, 200 μm.

(B) Immunofluorescence staining of ffEPSC-derived cardiomyocytes. Cardiomyocytes express neatly arranged sarcomeric α-actinin (SAA). Scale bar, 50 μm.

(C) The positive rate of cTnT in ffEPSC-derived cardiomyocytes determined by flow cytometric analysis.

(D and E) Representative bright and H&E staining images of the EHT on day 14. Scale bar, 50 μm.

(F) Immunofluorescence staining of the EHT constructed from ffEPSC-derived cardiomyocytes. The EHT shows neatly arranged sarcomeres and oriented nuclear arrangement. Scale bar, 20 μm.

EHTs with excellent functions constructed from ffEPSC-derived cardiomyocytes

EHTs begin self-assembling on the first day, forming cell connections from the center and gradually compacting. After two weeks of cultivation, the initial diameter of the EHTs decreases to about 100 μm. The cardiomyocytes are evenly distributed throughout the heart tissue without forming cell clusters. The well-developed sarcomeric structures fill the entire tissue and are oriented toward tissue stretch. EHTs can generate contractile forces at the mN level and have excellent conduction properties (Figure 3).

Figure 3.

Functional testing of EHTs

(A) Representative images of the EHT at its original state and 12% stretch.

(B) Representative contractile force wave in response to different stretch degrees.

(C) Schematic diagram of different analysis indicators of an EHT contractile’s single waveform signal.

(D) Statistics show that active force in 2-week EHT was constructed with ffEPSC-CMs during the progressive stretch of electrical stimulation (2 Hz). n = 3 for each group.

(E) Representative spatial maps of activation time and Ca²⁺ wave direction for EHTs stimulated with 1 Hz pacing and 10^-6 M isoproterenol (iso), demonstrating well-ordered tissue behavior.

(F and G) Schematic diagram and statistics of distributions toward propagating directions.

(H) Representative calcium transient traces of 2-week ffEPSC-CM EHT stimulated with 1 Hz pacing and 10^-6 M isoproterenol (iso).

(I–L) Quantitative analysis and statistics of conduction velocity (I), amplitude (J), rise time (K), and APD80 (L) of 2-week ffEPSC-CM EHT stimulated with 1Hz electrically and 10^-6 M isoproterenol (iso). n = 3 for each group. Data are represented as mean ± SEM.

Quantification and statistical analysis

Data were presented as mean ± SEM from at least three independent biological experiments. Statistical significance was determined using Student t test between 2 groups. In all cases, p-value < 0.05 was considered statistically significant. Statistical analysis was performed with GraphPad Prism (version 8.0.2).

Limitations

Although this approach offers a novel method for generating feeder-free extended pluripotent stem cells (ffEPSCs) and functionalized organized cardiac tissue, we are not sure whether it works smoothly for all ESC/iPSC lines. Here we have validated this approach with two ESC lines (HUES8, H9) and two iPSC lines (PGP1, WTC). Some adjustments might be required for other cell lines.

Troubleshooting

Problem 1

Excessive apoptosis during ffEPSC conversion (related to step 4).

Potential solution

Choose healthier and more suitable seed cells. Cultivate the cells in mTeSR1 medium for several generations, then select cells that form three-dimensional, well-defined colonies with transparent edges, no signs of differentiation, and excellent proliferative capacity for use as seed cells.

Increasing cell seeding density during passaging: We generally recommend increasing the cell seeding density during passaging. Due to inevitable cell death during transformation, varying cell states, and genetic backgrounds, the cell seeding density can be doubled if excessive cell death occurs.

Increasing Matrigel concentration: A range of 2%–3% Matrigel is recommended. A concentration of 3% or higher Matrigel helps reduce cell death. Additionally, at a 3% Matrigel concentration, the dome morphology of ffEPSCs is better. Therefore, a higher Matrigel concentration can help smoothly transition through the early stages of transformation.

Preventing mycoplasma and other contamination: Healthy cell states and environmental factors are crucial for ffEPSC conversion. It is essential to eliminate contamination interference and regularly test cells.

Problem 2

ffEPSC clones that are too large result in abnormal states (related to step 11).

Potential solution

Extend digestion time: We recommend using TrypLE for 4–5 min during passaging. However, the digestion time should be extended if ffEPSCs cannot be successfully dissociated into small clusters of 5–10 cells each. 8–10 min of TrypLE treatment is acceptable, as it helps disperse ffEPSCs into the appropriate size and maintain their proper dome shape.

Problem 3

Low cardiomyocyte differentiation efficiency (related to step 24).

Potential solution

Increase the ffEPSC seeding density. Improve the cell confluence at the beginning of differentiation. The confluence must be greater than 95%. Moreover, the differentiation could be started on the third day as alternative.

Problem 4

EHT function performance is not good enough (related to step 30).

Potential solution

When constructing tissue, adjust the ratio of myocardial cells. The higher and lower ratio is usually not conducive to the growth of EHTs. Make sure the cells are even when constructing tissue. During dynamic culture, adjust the angle and frequency of the shaker to allow the tissue to be fully exercised.

Resource availability

Lead contact

Further information and requests for resources and reagents can be provided by the lead contact, Dr. Donghui Zhang (dongh.zhang@hubu.edu.cn).

Technical contact

Requests for additional information about the protocol should be directed to Zhongjun Wan (wzj@stu.hubu.edu.cn).

Materials availability

This protocol did not generate new unique reagents.

Data and code availability

There are no additional datasets associated with this protocol.

Acknowledgments

We thank Drs. Jie Yang and Yan Qi and other laboratory members for their technical help and discussion. This work was supported by the National Key R&D Program of China (2021YFA1101902), the National Natural Science Foundation of China (no. 32171107 and 32350019), and the Natural Science Foundation of Hubei Province – Innovation Group project (2024AFA018).

Author contributions

Conceptualization, D.Z. and W.J.; investigation, Z.W., S.W., L.L., and R.Z.; writing – original draft, Z.W., S.W., L.L., and R.Z.; writing – review and editing, D.Z. and W.J.; funding acquisition, D.Z. and W.J.

Declaration of interests

W.J., R.Z., D.Z., L.L., and Z.W. have filed patents, respectively.